When observing an optically transparent microsamples including biological samples such as cells or tissues, there are some problems with a conventional optical imaging technique that it is very difficult to obtain clear morphological images for various intracellular organelles and substances in the sample and to measure spatial distribution of molecular species. This is because sufficient optical contrast can hardly be provided due to an insignificant difference between objects and background substances in the sample-light interaction. In other words, a specific microstructure to be observed in the sample or an extremely small quantity of substance is not distinguished from the background substances surrounding the circumference thereof.
To study biological phenomena and disease mechanisms in the cellular level by observing various metabolic substances and functioning intracellular organelles, an alternative optical imaging method has been proposed to overcome the limitations of general optical microscopes. Particularly, an optical technology for obtaining molecular images by detecting characteristic spectroscopic features unique to substances themselves has come into the spotlight. The Raman scattering spectroscopy is being widely used as a label-free method to analyze molecular vibrational fingerprints by making a specific molecule interact with a laser beam and allow for characteristic spectroscopic signals without help of molecule-selective markers. The Raman scattering spectroscopy is combined with an optical microscope system to obtain chemical images of microstructures in the sample.
FIG. 1 is a schematic diagram of molecular vibrational transitions illustrating the principle of a spontaneous Raman scattering signal generation.
Pixel data constituting a molecular image in a Raman microscope consist of spontaneous Raman spectra generated by scanning a laser beam of a single predetermined wavelength onto a sample. Photons of an incident laser beam at a fixed wavelength that do not cause resonance with molecular vibrational modes, generate red-shifted Raman spectral signals through inelastic scattering. Here, the energy difference between the incident photons and the Raman scattered photons corresponds to the energy of vibrational modes of the substance. That is, the Raman spectra obtained by collecting laser beams scattered by the sample contain information on a specific molecular vibrational mode of substance constituting the sample.
The aforementioned Raman microscope has both practical advantages and fundamental limitations. Since a single wavelength cw light source can be used in the Raman microscope regardless of a molecular vibrational frequency, there is an advantage in that the requirement for the excitation laser light source in the configuration of the Raman microscope is not so demanding, and its operation is relatively simple. Further, a laser light source having a short wavelength in the visible or UV region is used, which allows for obtaining microscopic images with high spatial resolution. On the other hand, the intensity of a Raman scattering signal containing molecular information is extremely weak, which gives rise to a fundamental problem that it usually takes a long integration time to obtain a microscopic image. Particularly, when dynamics of living biological samples are observed or the excitation laser power cannot be intense enough due to the optical damage threshold of the sample, such disadvantage becomes more serious.
A microscope based on the coherent anti-Stokes Raman scattering (CARS) spectroscopy can be a good alternative for overcoming the limitations of the conventional Raman microscope, which can dramatically improve the detection sensitivity and the frame rate in the chemical imaging. The CARS microscope is similar to the conventional Raman microscope in that the Raman scattering mechanism is exploited in detecting molecular vibrations. While spontaneous Raman scattering that is a linear optical process is used in the conventional Raman microscope, the CARS microscope is designed to use a kind of four-wave mixing process, namely, CARS. In the four-wave mixing, a nonlinear optical signal is generated by allowing three incident laser beams to interact with a sample.
FIG. 2 is a schematic diagram of molecular vibrational transitions illustrating the principle of CARS process.
The principle of the CARS microscopy is as follows. When two laser beams (a pump beam and a Stokes beam) having a frequency difference tuned to a Raman shift of specific molecules are incident on a sample, a harmonic oscillation of the molecules is resonantly induced, according to the phase of the beat waveform. If a third laser beam (a probe beam) enters the sample and interacts with such vibrating molecules, a strong coherent signal beam is generated in a specific direction through anti-Stokes Raman scattering process in which the wavelength of the signal beam is shortened after the interaction. Such nonlinear optical signal can be precisely mapped on a sample space at a high speed, thereby resulting in CARS microscopic images.
The most important advantage of the CARS microscope is to obtain selective molecular vibrational images like the Raman microscope but to provide a very high sensitivity and a very high speed in the image data acquisition. Since the CARS generates a signal beam at least 10,000 times stronger than the spontaneous Raman scattering even under the intensity-limiting condition for incident lasers which does not cause damages to the sample, a high-quality image having a high signal-to-noise ratio can be rapidly acquired. Since the CARS is a four-wave mixing process resulting from a third-order nonlinear optical response of a substance, the CARS signal can be enhanced proportional to the cube of the intensity of incident laser beams, which allows for nondestructive 3-dimensional imaging of the interior of a sample with high spatial resolution like a confocal laser scanning microscope. Since the CARS phenomenon is an optical parametric conversion process as well, no laser energy is deposited on the sample after laser interaction, thereby preventing the sample from being thermally damaged by a laser beam.
However, CARS spectroscopy has a major disadvantage in practice that the molecular selectivity and signal-to-noise ratio can be degraded by the non-resonant contribution in the third-order nonlinear susceptibility irrespective of molecular vibrational characteristics. Another technical disadvantage of the CARS spectroscopy compared with the Raman spectroscopy using a single wavelength light source is that the CARS spectroscopy could record molecular vibrational spectra by changing the wavelength of any one of two incident laser beams (a pump beam and a Stokes beam). That is, a laser light source generating a pump beam or a Stokes beam should have fast and stable wavelength tuning capability. Up to now, a picosecond/femtosecond laser light source suitable for biomedical CARS images hardly satisfies the required condition.
FIG. 3 is a schematic diagram of molecular vibrational transitions illustrating the principle of a 2-color multiplex CARS spectroscopy, and FIG. 4 is a schematic diagram illustrating spectral characteristics obtained by a multiplex CARS spectroscopy.
A multiplex CARS scheme may be used as a practical CARS implementation method for obtaining a broadband molecular vibrational spectrum. The multiplex CARS scheme does not employ a wavelength-tunable laser light source but employs a light source that simultaneously generates laser radiations with a broad wavelength bandwidth. Generally, a broadband laser beam is used to serve as a Stokes beam for generating broadband multiplex CARS signals. Since the generated CARS signal contains multiplexed spectral components, a monochromator or spectrometer for separating and detecting respective spectral signal components is usually employed in the 2-color multiplex CARS spectrometer.
For a broadband laser light source used to implement the 2-color multiplex CARS spectrometer, the output wavelength band must be longer than the wavelength of a laser beam that serves as a pump beam so as to serve as a Stokes beam, based on the principle of a CARS signal generation (see FIG. 3). When the aforementioned condition is satisfied, a multiplex CARS spectrum to be generated permits a spectral resolution defined by the linewidth of a pump laser beam at a fixed wavelength (see 4(b)). On the contrary to the aforementioned condition, if the broadband laser source operates in a shorter wavelength band, the broadband laser beam simultaneously serves as a pump beam and a probe beam. In turn, multiplex CARS spectral signals that are mixed in wavelength components by the spectral convolution result in to blur the original CARS spectrum (see FIG. 4(b)).
In principle, the combination of a broadband laser light source and a fixed wavelength laser source having a narrow linewidth, which are required to construct the 2-color multiplex CARS spectrometer, may be implemented in various ways. However, in the application of the multiplex CARS spectroscopy to biological samples, laser light sources for the 2-color multiplex CARS spectrometer should preferably operate in the near IR region with wavelength over 700 nm so as to avoid undesired laser-sample interactions other than the CARS process. Particularly, when observing turbid media such as biological tissues, the depth to which a laser beam is transmitted into the turbid media can be increased as the wavelength is lengthened. Therefore, selecting the wavelength of the laser light source is an important technical issue to be considered.
Generally, the Raman shift to be covered in the CARS spectroscopy lies in the range from 500 to 4000 cm−1. When the laser beam of a short wavelength, which serves as a pump beam, operates at a wavelength of over 700 nm, the broadband laser beam that serves as a Stokes beam must operate at a wavelength of over 950 nm. Practically, ultrashort pulse laser light sources capable of broadband lasing at these wavelengths are not easily available to date. As an alternative approach, there is a method using a super-continuum white-light beam generated from a photonic crystal fiber.
The super-continuum white-light beam may be generated by injecting a Ti:Sapphire femtosecond laser beam or neodimuim (Nd) gain medium modelocked picosecond laser beam into a photonic crystal fiber having a high nonlinearity. Here, the Ti:Sapphire femtosecond laser beam usually has its center wavelength near 800 nm where high power output can be achieved, and the Nd gain medium modelocked picosecond laser beam has a center wavelength of 1064 nm. A conversion of such laser light into broadband radiation can be used for a Stokes beam in the multiplex CARS spectroscopy. However, total conversion efficiency is often very low (below 5%), and it is technologically difficult to obtain a stable output spectrum having a uniform spectral shape. Fundamentally, the output power of an incident laser beam should be regulated due to the optical damage threshold of the photonic crystal fiber, and therefore, it is difficult to obtain a Stokes beam having a sufficient output required in the multiplex CARS spectroscopy. Consequently, if a Stokes beam having the aforementioned characteristics is employed, it takes a long time to obtain a multiplex CARS spectrum, and the signal-to-noise ratio of a spectrum is unwantedly low. Further, a careful spectrum normalization procedure is also necessary for quantitative measurements.
Therefore, an alternative scheme is required in order to use a broadband ultrashort pulse laser light source having a center wavelength near 800 nm which provides a stable and sufficient power output at the level of state-of-the-art technology. That is, it is required to develop an effective multiplex CARS spectroscopy, in which a broadband laser beam having a short wavelength is used as a pump beam, and a stable laser beam having a relatively long wavelength serves as a Stokes beam.